In the medical and analytical field, there are many situations where it is useful, even necessary, to detect interaction events between molecules and/or objects like antibodies, cells, bacteria, viruses and macromolecules. For example, to detect bacterial contamination, an antigen/antibody test is frequently used.
To determine an individual's blood group, an affinity test between antibodies and the individual's red blood cells is frequently used, thus enabling the blood group of said individual to be determined.
In the state of the art, there are “simple” methods that can be done when substances to be detected are present in large quantity. For example, this can be a method comprising a mixture of substances to be detected with affinity substances, i.e., substances that can interact with the substances to be detected. The interaction leads to the formation of a precipitate or aggregate visible to the naked eye which reveals the result. In this way, for example, it is possible to type a blood group by adding antibodies to a drop of blood and observing whether or not a red blood cell aggregate forms.
There are also methods that allow measuring the viscosity variations in a medium that occur when affinity substances come together in this medium.
For example, document U.S. Pat. No. 3,635,678 (Ref 1) describes a method in which a single macroscopic (much larger than a micron) steel ball immersed in the fluid is suspended by a set of magnets, the movement of the suspended ball being measured by an optical system. When the viscosity of the medium increases, the amplitude of the ball's movement decreases over time; the objective is to deduce the blood coagulation speed.
One major drawback of the technique described in this document is the complexity of its implementation, since a ball must be kept in suspension. Furthermore, the moving ball, because of its size and mass as described in the patent, can break weak interactions that cause the viscosity variation and impede their detection. The method is not very sensitive and is limited to detecting relatively large viscosity variations. Moreover, to read the results, complex systems and reagents must be used.
Another system is described in document WO 01/86255 (Ref 2) comprising a microscope with which it is possible to observe movement of a particle suspended in a liquid. The viscosity of the liquid is deduced by phase shift of the second harmonic of the signal obtained from observing the particle in suspension in a liquid with the microscope.
Another variant of this approach is described in document EP 1,544,596 (Ref 3), which comprises oscillation of a magnetizable particle in a magnetic field to generate a signal and therefore obtain a result.
One of the major drawbacks of these methods is the complexity of their implementation and of monitoring the oscillation of a particle activated by a force applied in a periodic and controlled manner. The complex, costly and difficult to configure instrumentation, on the one hand, to start and maintain the oscillation of the particle and, on the other hand, to observe the periodic movement of the particle.
Moreover, these methods only allow measuring the changes in viscosity that must take place in the oscillation zone of the suspended particle. Moreover, the viscosity changes must be substantial to be detected. Thus, it is necessary to have solutions that contain a large quantity of affinity substances. These methods are therefore not very sensitive and do not permit satisfactory precision to be obtained for methods using antibodies, for example, or other specifically directed affinity substances to detect or assay.
Another commonly used method to detect affinity reactions is the ELISA technique and its many variations. It consists of:                attaching onto a substrate a first antibody having an affinity with a substance,        contacting the substance to be detected with the first antibody attached to its substrate for a given duration,        rinsing away the substance that did not react with the antibody,        contacting the substance bound to the antibody attached to the surface and a second antibody with an affinity for said substance,        rinsing away the antibodies that did not react with the substance, and        detecting the presence of the second antibody bound to the substance bound to the first antibody bound to the substrate.        
Most often, the second antibody is bound to a marker that is directly detectable by a physical method, for example a fluorescent or magnetic marker, or to a marker bound to an enzyme, for example alkaline phosphatase or horseradish peroxidase, in order to detect the product of the reaction catalyzed by the enzyme from an appropriate substrate.
This method has several drawbacks, for example it requires many controlled washes, reducing its sensitivity. It also comprises many steps of manipulation by a person skilled in the art: conducting at least two affinity reactions, required marking of the second antibody with at least one sensitive and sophisticated marker, and using a complex device that is difficult to configure to detect and quantify the signal emitted by the marker. This method is therefore time-consuming and expensive, and requires complex instrumentation for its implementation.
Other methods have been developed to detect an affinity reaction that require only a single antibody, for example techniques based on surface plasmon resonance, piezoelectric balances, or even simple observation by microscope for some substances permitting it. To do this, the antibody is attached to a substrate appropriate for the detection method, the substance is contacted for a given time with the antibody attached to the substrate, and then the reading is done directly or after rinsing to remove the substances that did not have an affinity reaction with the antibodies.
These methods have many drawbacks; for example, the instrument required to do the reading is sophisticated and expensive. Moreover, they require special substrates for attaching said antibodies. Furthermore, they require a large number of steps and depend on the quality and quantity of antibodies attached.
In the above-mentioned methods, it is therefore essential to attach the antibody or any other affinity substance to a substrate. Affinity surfaces are known in the state of the art. Such surfaces may be obtained, for example, by “molecular molding”.
It is also known that the above-named methods can be implemented by attaching the substance that one wishes to detect onto a substrate in a non-specific way. The substance thus attached will be contacted with an antibody or any other suitable affinity substance, possibly labelled, according to the detection method, and then possibly rinsed before being read.
However, these variants have many drawbacks. In particular, it is necessary to bind the substance to be detected onto the substrate, which is not possible in a universal or specific manner. Moreover, this attachment can change the structure of the substance attached. This structural change can alter detection sensitivity and/or specificity, in particular when antibodies are used. Moreover, the attachment can lead to obtaining false negative and/or false positive results. Moreover, the attachment may be different from one implementation to another, so the results thus obtained may not be reproducible. Moreover, complex and expensive devices are required for detection.
There is a thus real need to find a method for detecting molecular interactions alleviating these deficiencies, drawbacks and obstacles of the prior art, in particular, a method that improves the sensitivity of molecular interaction detection and reduces the costs of implementation, a method whose implementation is simple and which provides fast, reliable and reproducible results.